Recombinant expression of aryl hydrocarbon receptor for quantitative ligand-binding analysis.
Recombinant expression of the aryl hydrocarbon receptor (AhR) yields small amounts of ligand-binding-competent AhR. Therefore, Spodoptera frugiperda (Sf9) cells and baculovirus have been evaluated for high-level and functional expression of AhR. Rat and human AhR were expressed as soluble protein in significant amounts. Expression of ligand-binding-competent AhR was sensitive to the protein concentration of Sf9 extract, and coexpression of the chaperone p23 failed to affect the yield of functional ligand-binding AhR. The expression system yielded high levels of functional protein, with the ligand-binding capacity (Bmax) typically 20-fold higher than that obtained with rat liver cytosol. Quantitative estimates of the ligand-binding affinity of human and rat AhR were obtained; the Kd for recombinant rat AhR was indistinguishable from that of native rat AhR, thereby validating the expression system as a faithful model for native AhR. The human AhR bound TCDD with significantly lower affinity than the rat AhR. These findings demonstrate high-level expression of ligand-binding-competent AhR, and sufficient AhR for quantitative analysis of ligand binding.
Fan
Ming Qi
MQ
School of Biology, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
Bell
Alex R
AR
Bell
David R
DR
Clode
Sally
S
Fernandes
Alwyn
A
Foster
Paul M D
PM
Fry
Jeffrey R
JR
Jiang
Tao
T
Loizou
George
G
MacNicoll
Alan
A
Miller
Brian G
BG
Rose
Martin
M
Shaikh-Omar
Osama
O
Tran
Lang
L
White
Shaun
S
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D006801:Humans; D008024:Ligands; D051381:Rats; D017208:Rats, Wistar; D018336:Receptors, Aryl Hydrocarbon / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
D008024:Ligands; D018336:Receptors, Aryl Hydrocarbon; D011994:Recombinant Proteins